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Avian Dis. Author manuscript; available in PMC 2016 August 29. Published in final edited form as: Avian Dis. 2016 May ; 60(1 Suppl): 359–364. doi:10.1637/11130-050715-Reg.
Prevalence and diversity of low pathogenicity avian influenza viruses in wild birds in Guatemala, 2010-2013 Ana S. Gonzalez-Reiche1,2,3, Maria L. Müller2, Lucía Ortiz2, Celia Cordón-Rosales2, and Daniel R. Perez1 1Department
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of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America. 953 College Station Road, Athens, GA 30602, U.S.A 2Laboratorio
de Virus Zoonóticos, Centro de Estudios en Salud, Universidad del Valle de Guatemala (CES-UVG). 18 Ave. 11-95, Zona 15 V.H.3. Guatemala, 01015, Guatemala
3Department
of Veterinary Medicine, University of Maryland College Park, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, United States of America. 8075 Greenmead Drive, College Park, MD, 20742, U.S.A
Summary
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Waterfowl species are known to harbor the greatest diversity of low pathogenicity influenza A virus (LPAIV) subtypes and are recognized as their main natural reservoir. In Guatemala there is evidence of circulation of LPAIV in wild ducks, however the bird species contributing to viral diversity during the winter migration in Central America are unknown. In this study, samples obtained from 1,250 hunter-killed birds from 22 different species were collected on the Pacific coast of Guatemala during three winter migration seasons between 2010 and 2013. Prevalence of LPAIV detected by real-time reverse-transcriptase polymerase chain reaction was 38.2%, 23.5% and 24.7% in the 2010-11, 2011-12, and 2012-13 seasons respectively. The highest virus prevalence was detected in the northern shoveler (Anas clypeata), followed by the blue-winged teal (Anas discors). The majority of positive samples and viral isolates were obtained from the blue-winged teal. Analysis of LPAIV prevalence over time in this species indicated a decreasing trend in monthly prevalence within a migration season. Sixty-eight viruses were isolated and 9 HA and 7 NA subtypes were identified in 19 subtype combinations. In 2012-13, the most prevalent subtype was H14, a subtype identified for the first time in the western hemisphere in 2010. The results from this study represent the most detailed description available to date of LPAIV circulation in Central America.
Resumen Las aves acuáticas albergan la mayor diversidad de los virus de Influenza Aviar (IA) en la naturaleza, siendo reconocidas como su principal reservorio. En Guatemala, se ha documentado que los virus de IA circulan en aves acuáticas silvestres, sin embargo poco se sabe de cuáles son las especies que contribuyen a la diversidad de los virus durante la época migratoria. En este estudio se colectaron 1.250 muestras de 22 especies diferentes de aves cazadas en la costa del
Corresponding authors: Ana S. Gonzalez-Reiche, [email protected]; Daniel R. Perez, [email protected].
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Pacífico de Guatemala durante tres estaciones migratorias, entre 2010 y 2013. La prevalencia de IA detectada por transcripción-inversa y reacción en cadena de la polimerasa en tiempo real fue 38.2%, 23.5% y 24.7% durante las estaciones de 2010-11, 2011-12 y 2012-13, respectivamente. La mayor prevalencia viral se observó en el pato cuchara (Anas clypeata), seguido del pato aliazul (Anas discors). Sin embargo, la mayoría de muestras positivas y aislados virales fueron obtenidos del pato aliazul. A través del análisis temporal de la prevalencia en esta especie, se observó que la prevalencia de IA disminuye a lo largo de la migración. Se aislaron sesenta y ocho virus de 9 subtipos de HA y 7 subtipos de NA en 19 combinaciones diferentes. Durante la estación migratoria de 2012-13 el subtipo más detectado fue el H14, un subtipo identificado por primera vez en el hemisferio occidental en 2010. A la fecha, los resultados de este estudio representan la única descripción a detalle de la circulación IA de baja patogenicidad reportados para Centro América.
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Keywords Avian influenza; Guatemala; Central America; surveillance; wild birds; prevalence; subtype diversity
Introduction
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Despite the increased awareness of the role of wild birds in the spread of avian influenza and the need to expand global efforts for surveillance of influenza A viruses (IAV), there are still significant knowledge gaps in the ecology of IAV. This is particularly the case with respect to the viruses that circulate in Central and South America (13). In 2012, an IAV outbreak caused by a highly pathogenic avian influenza virus (HPAIV) H7N3 strain of wild bird origin in Mexico resulted in significant economic losses for one of the biggest egg and poultry producers of Latin America (6, 23). Most recently the introduction of highly pathogenic H5 viruses of Eurasian origin (22, 26) raises concerns about further virus spreading across the region, with potentially devastating consequences for the developing countries in the Americas.
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In North America, mallards (Anas platyrhynchos) and northern shovelers (Anas clypeata) are two of the duck species with the highest prevalence of influenza A virus (IAV) (11, 19, 20, 43). In addition, the blue-winged teal (Anas discors) has been observed to harbor high diversity of virus subtypes in wintering grounds, in particular in locations across the Mississippi and Central migration flyways (7, 8, 16, 37, 43). Studies in wintering grounds in North America and Europe suggest that viral prevalence in waterfowl species tend to be low in comparison to the levels generally observed at the breeding grounds prior to the onset of autumn migration. The number of virus infections decreases over time in the population during an annual migration cycle that concludes with the spring migration, as birds return from the wintering grounds (15, 19, 30, 31). In Guatemala, we have previously reported evidence of circulation of IAV from the North American lineage in wild ducks (12); however the main bird species that contribute to viral diversity during and between migration seasons remains poorly understood. Convergence of multiple flyways into a reduced geographical area, distinguishes the wintering grounds of Avian Dis. Author manuscript; available in PMC 2016 August 29.
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Central America from those in North America (32). In Europe, IAV surveillance in wild birds in a location where multiple flyways overlap, provided evidence of increased gene flow between host populations from different geographical regions, resulting in high diversity of locally circulating viruses (28). Similarly, congregation of bird populations from multiple migration flyways into a geographical bottleneck in Central America may provide unique conditions for virus reassortment during the winter migration. In this study, samples from hunter-killed waterfowl were collected on the Pacific coast of Guatemala during three consecutive winter migrations between 2010 and 2013. We estimated prevalence values for different bird species and compared subtype diversity among different seasons. In addition, we analyzed the patterns of IAV prevalence during the winter migration for blue-winged teal, the most abundantly sampled bird species, in order to characterize the dynamics of IAV circulation in wintering grounds in Central America.
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Methods Sample collection
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Samples were collected from hunter-killed ducks during the winter migration season from 2010 to 2013 in the villages of El Pumpo in the department of Santa Rosa, Pasaco in the department of Jutiapa and in La Gomera in the department of Escuintla. Sampling sites and tracheal and cloacal swab collection methods from birds has been as previously described (12). Permits for sampling different bird species at the different sampling sites were obtained from the Center for Conservation Studies (CECON) and the National Council of Protected Areas (CONAP). Sampling of hunter-killed birds was exempt from animal use and care regulations from the Institutional Animal Use and Care Committees of the University of Maryland and the Universidad del Valle de Guatemala. Virus detection All samples were tested for the presence of IAV RNA by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) (42). The details of the methods for RNA extraction, molecular testing and virus isolation have been described elsewhere (12). Only IAV positive samples by rRT-PCR were tested for virus isolation. The subtype of all viral isolates was identified by partial sequencing of the HA and NA genes with universal primers (21, 35).
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Prevalence of IAV was estimated for each bird species as the total number of rRT-PCR positives divided by the total number of individuals from the same species tested by rRTPCR. Confidence intervals were computed for prevalence values at the 95% confidence level when n>30. Differences in prevalence among duck species, and seasons (2010-11, 2011-12 and 2012-13) were analyzed with χ2 test at the 95% confidence level; only species with n>5 per sampling point (i.e. each collection date) were included in this analysis (blue-winged teal and northern shoveler). The calculations were done in GraphPad Prism v.6.0 (La Jolla, CA, www.graphpad.com). The variable location was excluded from the analysis as not all locations were uniformly sampled during all seasons. In addition, differences in monthly prevalence were analyzed for blue-winged teals with χ2 test at the 95% confidence level. The number of samples for the northern shoveler was not enough to do this analysis. For all
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analyses, a two-sided alternative hypothesis was assumed with p-values 30 have been reported (27, 29). In this study, CT-values for all rRT-PCR positive samples ranged from 19 to 41 with a median of 34 (data not shown). For samples positive for virus isolation the CT-values ranged between 19 and 38 with a median of 31. In addition, selection of the strains that are able to replicate in embryonating chicken eggs (ECE) during virus isolation may also explain the differences in prevalence estimates obtained with each method. Higher prevalence estimates based on rRTPCR data when compared to virus isolation are in agreement with other similar studies (27, 39). An increase in rRT-PCR virus prevalence compared to previous years was observed previously during the 2009-10 (12). The limited sample size may have influenced the unexpectedly high prevalence estimates obtained in 2010 (12) and during the first season of the present study. During the subsequent seasons (2011-12 and 2012-13) the number of samples was larger (n>500), however the estimated prevalence was still high in comparison to estimates reported for blue-winged teals in North America (31). The high diversity of cocirculating subtypes and subtype combinations detected may help explain this observation. High diversity of AIV subtypes have been observed in wintering grounds in California and Texas (7, 8, 19), with comparable virus isolation rates.
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During the 2010-11 and 2011-12 seasons a decreasing trend in prevalence was observed towards the end of the migration, supporting observations from previous years in Guatemala and similar to patterns of prevalence observed in wintering grounds in the south of the US (7, 8, 12, 19, 31). This decrease is likely explained by the accumulation of population immunity (as the number of seroconverted birds increases) resulting in a reduced number of susceptible individuals, not only to circulating subtypes but to other viruses from related genetic clades (25). During 2012-13 the pattern of IAV detection over time was different and the prevalence of IAV in January two-times higher in comparison to previous years (Figure 1). A plausible explanation could be the introduction and a potential outbreak of H14, the most prevalent HA subtype detected during that season. There is no evidence of circulation of H14 viruses in the western hemisphere prior to 2010 (4, 10). In January 2013, 4 of the 5 virus isolates obtained from blue-winged teals that month were from the H14 subtype. Current reports indicate that isolation of H14 viruses has been sporadic in North America (9, 10). It is not clear if the H14 virus persisted in wintering grounds in Guatemala after its first detection in 2011, or if it was re-introduced during the following season. In addition to the H14, other prevalent subtypes found in this study include the H3 and the H4, both of which are commonly isolated in North America (24). However, these subtypes were found in noncommonly isolated combinations such as H1N3 (the second most prevalent subtype in 2011-12), H3N3 and the H4N3. The H6 subtype, also prevalent in North America (2, 19),
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was not found in this study. Maintenance of virus diversity by resident duck populations has been observed in other studies (17, 18), and this diversity may be amplified upon arrival of migrants resulting in epizootic events (45). Either of these possibilities is supported by isolation of other rare subtypes (including the H14) and subtype combinations at the wintering grounds in Guatemala.
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Among other species that tested positive for IAV were two white-winged doves. The number of samples collected from doves in this study was limited and no isolates were obtained from the rRT-PCR positive samples. There is natural and experimental evidence that doves and pigeons are susceptible to infection with IAV, including the H7N9 subtype from Asia (1). Although their role in IAV transmission remains unclear, association with habitats where waterfowl species are abundant, such as wetlands, may increase the probability of exposure to IAV in terrestrial birds. Nonetheless, the number of natural infections observed has been limited, suggesting that these species may solely act as incidental hosts.
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In summary, we detected a wide diversity of virus subtypes in ducks during the wintering season in Guatemala. The diversity of circulating viruses seems to vary between years and overall virus prevalence seems to decrease at the end of the migration season at the studied locations. Our findings are supported by previous observations at the wintering grounds in Guatemala and other locations. The emergence of the H14 subtype in blue-winged teals (4, 10, 33, 36), and its high levels of detection during the 2012-13 season, provides further evidence that the wintering grounds in Central America may serve as places where virus variants with limited circulation may be amplified upon arrival of yearly migrants. Although additional bird species need to be investigated, the high relative abundance of the bluewinged teals in comparison to other duck species in Central America, the particular behavior of this long-distance migrant (3, 34, 38), and the diversity of viruses found in these ducks makes them a candidate species for targeted IAV surveillance in the Neotropics. We recognize that surveillance in other species, characterization of viruses that circulate in resident bird populations (during and in between migrations), and incorporation of more systematic sampling methodologies, (including the use of geo-transmitters), are needed to better understand the ecology of IAV in this region; however, as we cannot longer ignore that Eurasian HPAI H5 viruses have been introduced to the western hemisphere, we think that sampling of hunted birds is a cost effective strategy that could be replicated in neighboring countries from the region that, like Guatemala, have limited resources to establish long term disease surveillance.
Acknowledgements Author Manuscript
The authors want to thank national regulatory agencies (CONAP and CECON) and licensed sport-hunters for allowing sample collection from birds in Guatemala. To Jorge Paniagua, Silvia Ramirez, Silvia Sosa, Carmen Yoc, Oscar de Leon, Adan Real, Johanna Lavigne, Diego Lopez for their assistance in sample collection and processing, data management, and administrative support. To Cheryl Nichols for English editing of this manuscript. This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) Center for Research on Influenza Pathogenesis (CRIP) contracts No. HHSN266200700010C and HHSN272201400008C.
Abbreviations AIV
Avian influenza virus
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HA (H)
hemagglutinin
NA (N)
neuraminidase
LPAIV
Low pathogenicity avian influenza virus
HPAIV
Highly pathogenic avian influenza virus
rRT-PCR
real-time reverse-transcriptase polymerase chain reaction
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Figure 1.
Temporal distribution of IAV prevalence in blue-winged teals in Guatemala, 2010-2013
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Figure 2.
Virus subtypes (HA and NA) by month obtained from wintering waterfowl in Guatemala between 2010 and 2013.
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Avian Dis. Author manuscript; available in PMC 2016 August 29. 1(50)
2
Total
102
129(23.5)
(0) 39(38.2)
2
Catoptrophorus semipalmatus (Willet)
Scolopacidae
1(50)
Fulica americana (American Coot)
Rallidae
Eudocimus albus (White Ibis)
0(0) 1(25)
Phalacrocorax brasilianus (Neotropic Cormorant)
Phalacrocoracidae
(0)
0(0)
Pelecanus erythrorhynchos (American White Pelican)
Limnodromus scolopaceus (Long-billed Dowitcher)
0(0)
Zenaida macroura (Mourning Dove)
Pelecanidae
550
1
1
4
1
1
2
148(24.7)
0(0)
0(0)
0(0)
0(0)
2(40)
8
1
1
1
2
1
1(100) 135(24.5)
0(0)
0(0)
Columba flavirostris (Red-billed Pigeon)
Columbidae
16 434
Zenaida asiatica (White-winged Dove)
0(0)
Mycteria americana (Wood Stork)
Ciconiidae
Threskiornithidae
0(0)
Positive (%)
Streptopelia decaocta (Eurasian Collared-Dove)
0(0)
(0)
2(100)
Dendrocygna bicolor (Fulvous Whistling-Duck) 2
(0)
1(50)
2
0(0)
(0)
Dendrocygna autumnalis (Black-bellied WhistlingDuck)
7
1(6.3) 99(22.8)
Cairina moschata (Muscovy Duck)
Anas sp. (Dabbling duck)
1 82
0(0)
4(57.1)
Anas discors (Blue-winged Teal)
Aythya affinis (Lesser Scaup)
0(0) 29(35.4)
Anas crecca (Green-winged Teal)
74
2
Total
8(29.6)
28(37.8)
0(0)
Positive (%)
2(28.6) 4
Total
Anas clypeata (Northern Shoveler) 1(25)
Positive (%)
598
1
5
2
1
1
550
1
27
7
3
Total
2012-2013
Anas americana (American Wigeon)
Anas acuta (Northern Pintail)
Species (Common name)
2011-2012
Oxyura jamaicensis (Ruddy Duck)
Anatidae
Family
2010-2011
316(25.3)
1(25)
0(0)
1(50)
1(25)
0(0)
0(0)
0(0)
2(15.4)
0(0)
0(0)
0(0)
0(0)
2(100)
1(50)
0(0)
0(0)
4(57.1)
263(24.7)
2(11.1)
37(35.2)
2(28.6)
0(0)
Positive (%)
Total
1250
4
1
2
4
1
1
2
13
2
1
1
2
2
2
2
2
7
1066
18
105
7
5
Total
Percentage prevalence for waterfowl species sampled during the wintering migration seasons in Guatemala, 2010-2013
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Table 1 Gonzalez-Reiche et al. Page 13
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Author Manuscript H5N3
Anas crecca (Green-winged teal)
Anas discors (Blue-winged teal)
1
-
Total
-
Anas clypeata (Northern shoveler)
2010
Anas americana (American wigeon)
Species
H11N3 H11N9
H4N8
21
H14N5 -
H4N6
H4N3 (2)
21
H4N2 (4)
H4N2 (2)
H14N3 (7)
H12N5 H14N3 (2)
H3N8
12
H11N3
H5N3
H3N3 (2)
H1N3 (3)
H3N3
H3N8 (2)
H14N6
-
H11N3
-
2011
H3N2 (10)
-
-
-
-
2010
H3N2 (4)
-
-
-
2012
December
H1N3 (3)
-
H1N3
-
2011
November
5
H4N2
H3N2
H2N3
-
H4N2
H4N2
2012
1
-
-
H7N3
-
2012
7
H5N3
H14N4 (2)
H14N3 (2)
H7N9
H3N8
-
2013
January
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Subtypes of IAV isolated during the wintering season in Guatemala, 2010-2013
68
61
1
5
1
Total
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Table 2 Gonzalez-Reiche et al. Page 14
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Avian Dis. Author manuscript; available in PMC 2016 August 29. Published in final edited form as: Avian Dis. 2016 May ; 60(1 Suppl): 359–364. doi:10.1637/11130-050715-Reg.
Prevalence and diversity of low pathogenicity avian influenza viruses in wild birds in Guatemala, 2010-2013 Ana S. Gonzalez-Reiche1,2,3, Maria L. Müller2, Lucía Ortiz2, Celia Cordón-Rosales2, and Daniel R. Perez1 1Department
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of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America. 953 College Station Road, Athens, GA 30602, U.S.A 2Laboratorio
de Virus Zoonóticos, Centro de Estudios en Salud, Universidad del Valle de Guatemala (CES-UVG). 18 Ave. 11-95, Zona 15 V.H.3. Guatemala, 01015, Guatemala
3Department
of Veterinary Medicine, University of Maryland College Park, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, United States of America. 8075 Greenmead Drive, College Park, MD, 20742, U.S.A
Summary
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Waterfowl species are known to harbor the greatest diversity of low pathogenicity influenza A virus (LPAIV) subtypes and are recognized as their main natural reservoir. In Guatemala there is evidence of circulation of LPAIV in wild ducks, however the bird species contributing to viral diversity during the winter migration in Central America are unknown. In this study, samples obtained from 1,250 hunter-killed birds from 22 different species were collected on the Pacific coast of Guatemala during three winter migration seasons between 2010 and 2013. Prevalence of LPAIV detected by real-time reverse-transcriptase polymerase chain reaction was 38.2%, 23.5% and 24.7% in the 2010-11, 2011-12, and 2012-13 seasons respectively. The highest virus prevalence was detected in the northern shoveler (Anas clypeata), followed by the blue-winged teal (Anas discors). The majority of positive samples and viral isolates were obtained from the blue-winged teal. Analysis of LPAIV prevalence over time in this species indicated a decreasing trend in monthly prevalence within a migration season. Sixty-eight viruses were isolated and 9 HA and 7 NA subtypes were identified in 19 subtype combinations. In 2012-13, the most prevalent subtype was H14, a subtype identified for the first time in the western hemisphere in 2010. The results from this study represent the most detailed description available to date of LPAIV circulation in Central America.
Resumen Las aves acuáticas albergan la mayor diversidad de los virus de Influenza Aviar (IA) en la naturaleza, siendo reconocidas como su principal reservorio. En Guatemala, se ha documentado que los virus de IA circulan en aves acuáticas silvestres, sin embargo poco se sabe de cuáles son las especies que contribuyen a la diversidad de los virus durante la época migratoria. En este estudio se colectaron 1.250 muestras de 22 especies diferentes de aves cazadas en la costa del
Corresponding authors: Ana S. Gonzalez-Reiche, [email protected]; Daniel R. Perez, [email protected].
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Pacífico de Guatemala durante tres estaciones migratorias, entre 2010 y 2013. La prevalencia de IA detectada por transcripción-inversa y reacción en cadena de la polimerasa en tiempo real fue 38.2%, 23.5% y 24.7% durante las estaciones de 2010-11, 2011-12 y 2012-13, respectivamente. La mayor prevalencia viral se observó en el pato cuchara (Anas clypeata), seguido del pato aliazul (Anas discors). Sin embargo, la mayoría de muestras positivas y aislados virales fueron obtenidos del pato aliazul. A través del análisis temporal de la prevalencia en esta especie, se observó que la prevalencia de IA disminuye a lo largo de la migración. Se aislaron sesenta y ocho virus de 9 subtipos de HA y 7 subtipos de NA en 19 combinaciones diferentes. Durante la estación migratoria de 2012-13 el subtipo más detectado fue el H14, un subtipo identificado por primera vez en el hemisferio occidental en 2010. A la fecha, los resultados de este estudio representan la única descripción a detalle de la circulación IA de baja patogenicidad reportados para Centro América.
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Keywords Avian influenza; Guatemala; Central America; surveillance; wild birds; prevalence; subtype diversity
Introduction
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Despite the increased awareness of the role of wild birds in the spread of avian influenza and the need to expand global efforts for surveillance of influenza A viruses (IAV), there are still significant knowledge gaps in the ecology of IAV. This is particularly the case with respect to the viruses that circulate in Central and South America (13). In 2012, an IAV outbreak caused by a highly pathogenic avian influenza virus (HPAIV) H7N3 strain of wild bird origin in Mexico resulted in significant economic losses for one of the biggest egg and poultry producers of Latin America (6, 23). Most recently the introduction of highly pathogenic H5 viruses of Eurasian origin (22, 26) raises concerns about further virus spreading across the region, with potentially devastating consequences for the developing countries in the Americas.
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In North America, mallards (Anas platyrhynchos) and northern shovelers (Anas clypeata) are two of the duck species with the highest prevalence of influenza A virus (IAV) (11, 19, 20, 43). In addition, the blue-winged teal (Anas discors) has been observed to harbor high diversity of virus subtypes in wintering grounds, in particular in locations across the Mississippi and Central migration flyways (7, 8, 16, 37, 43). Studies in wintering grounds in North America and Europe suggest that viral prevalence in waterfowl species tend to be low in comparison to the levels generally observed at the breeding grounds prior to the onset of autumn migration. The number of virus infections decreases over time in the population during an annual migration cycle that concludes with the spring migration, as birds return from the wintering grounds (15, 19, 30, 31). In Guatemala, we have previously reported evidence of circulation of IAV from the North American lineage in wild ducks (12); however the main bird species that contribute to viral diversity during and between migration seasons remains poorly understood. Convergence of multiple flyways into a reduced geographical area, distinguishes the wintering grounds of Avian Dis. Author manuscript; available in PMC 2016 August 29.
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Central America from those in North America (32). In Europe, IAV surveillance in wild birds in a location where multiple flyways overlap, provided evidence of increased gene flow between host populations from different geographical regions, resulting in high diversity of locally circulating viruses (28). Similarly, congregation of bird populations from multiple migration flyways into a geographical bottleneck in Central America may provide unique conditions for virus reassortment during the winter migration. In this study, samples from hunter-killed waterfowl were collected on the Pacific coast of Guatemala during three consecutive winter migrations between 2010 and 2013. We estimated prevalence values for different bird species and compared subtype diversity among different seasons. In addition, we analyzed the patterns of IAV prevalence during the winter migration for blue-winged teal, the most abundantly sampled bird species, in order to characterize the dynamics of IAV circulation in wintering grounds in Central America.
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Methods Sample collection
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Samples were collected from hunter-killed ducks during the winter migration season from 2010 to 2013 in the villages of El Pumpo in the department of Santa Rosa, Pasaco in the department of Jutiapa and in La Gomera in the department of Escuintla. Sampling sites and tracheal and cloacal swab collection methods from birds has been as previously described (12). Permits for sampling different bird species at the different sampling sites were obtained from the Center for Conservation Studies (CECON) and the National Council of Protected Areas (CONAP). Sampling of hunter-killed birds was exempt from animal use and care regulations from the Institutional Animal Use and Care Committees of the University of Maryland and the Universidad del Valle de Guatemala. Virus detection All samples were tested for the presence of IAV RNA by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) (42). The details of the methods for RNA extraction, molecular testing and virus isolation have been described elsewhere (12). Only IAV positive samples by rRT-PCR were tested for virus isolation. The subtype of all viral isolates was identified by partial sequencing of the HA and NA genes with universal primers (21, 35).
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Prevalence of IAV was estimated for each bird species as the total number of rRT-PCR positives divided by the total number of individuals from the same species tested by rRTPCR. Confidence intervals were computed for prevalence values at the 95% confidence level when n>30. Differences in prevalence among duck species, and seasons (2010-11, 2011-12 and 2012-13) were analyzed with χ2 test at the 95% confidence level; only species with n>5 per sampling point (i.e. each collection date) were included in this analysis (blue-winged teal and northern shoveler). The calculations were done in GraphPad Prism v.6.0 (La Jolla, CA, www.graphpad.com). The variable location was excluded from the analysis as not all locations were uniformly sampled during all seasons. In addition, differences in monthly prevalence were analyzed for blue-winged teals with χ2 test at the 95% confidence level. The number of samples for the northern shoveler was not enough to do this analysis. For all
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analyses, a two-sided alternative hypothesis was assumed with p-values 30 have been reported (27, 29). In this study, CT-values for all rRT-PCR positive samples ranged from 19 to 41 with a median of 34 (data not shown). For samples positive for virus isolation the CT-values ranged between 19 and 38 with a median of 31. In addition, selection of the strains that are able to replicate in embryonating chicken eggs (ECE) during virus isolation may also explain the differences in prevalence estimates obtained with each method. Higher prevalence estimates based on rRTPCR data when compared to virus isolation are in agreement with other similar studies (27, 39). An increase in rRT-PCR virus prevalence compared to previous years was observed previously during the 2009-10 (12). The limited sample size may have influenced the unexpectedly high prevalence estimates obtained in 2010 (12) and during the first season of the present study. During the subsequent seasons (2011-12 and 2012-13) the number of samples was larger (n>500), however the estimated prevalence was still high in comparison to estimates reported for blue-winged teals in North America (31). The high diversity of cocirculating subtypes and subtype combinations detected may help explain this observation. High diversity of AIV subtypes have been observed in wintering grounds in California and Texas (7, 8, 19), with comparable virus isolation rates.
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During the 2010-11 and 2011-12 seasons a decreasing trend in prevalence was observed towards the end of the migration, supporting observations from previous years in Guatemala and similar to patterns of prevalence observed in wintering grounds in the south of the US (7, 8, 12, 19, 31). This decrease is likely explained by the accumulation of population immunity (as the number of seroconverted birds increases) resulting in a reduced number of susceptible individuals, not only to circulating subtypes but to other viruses from related genetic clades (25). During 2012-13 the pattern of IAV detection over time was different and the prevalence of IAV in January two-times higher in comparison to previous years (Figure 1). A plausible explanation could be the introduction and a potential outbreak of H14, the most prevalent HA subtype detected during that season. There is no evidence of circulation of H14 viruses in the western hemisphere prior to 2010 (4, 10). In January 2013, 4 of the 5 virus isolates obtained from blue-winged teals that month were from the H14 subtype. Current reports indicate that isolation of H14 viruses has been sporadic in North America (9, 10). It is not clear if the H14 virus persisted in wintering grounds in Guatemala after its first detection in 2011, or if it was re-introduced during the following season. In addition to the H14, other prevalent subtypes found in this study include the H3 and the H4, both of which are commonly isolated in North America (24). However, these subtypes were found in noncommonly isolated combinations such as H1N3 (the second most prevalent subtype in 2011-12), H3N3 and the H4N3. The H6 subtype, also prevalent in North America (2, 19),
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was not found in this study. Maintenance of virus diversity by resident duck populations has been observed in other studies (17, 18), and this diversity may be amplified upon arrival of migrants resulting in epizootic events (45). Either of these possibilities is supported by isolation of other rare subtypes (including the H14) and subtype combinations at the wintering grounds in Guatemala.
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Among other species that tested positive for IAV were two white-winged doves. The number of samples collected from doves in this study was limited and no isolates were obtained from the rRT-PCR positive samples. There is natural and experimental evidence that doves and pigeons are susceptible to infection with IAV, including the H7N9 subtype from Asia (1). Although their role in IAV transmission remains unclear, association with habitats where waterfowl species are abundant, such as wetlands, may increase the probability of exposure to IAV in terrestrial birds. Nonetheless, the number of natural infections observed has been limited, suggesting that these species may solely act as incidental hosts.
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In summary, we detected a wide diversity of virus subtypes in ducks during the wintering season in Guatemala. The diversity of circulating viruses seems to vary between years and overall virus prevalence seems to decrease at the end of the migration season at the studied locations. Our findings are supported by previous observations at the wintering grounds in Guatemala and other locations. The emergence of the H14 subtype in blue-winged teals (4, 10, 33, 36), and its high levels of detection during the 2012-13 season, provides further evidence that the wintering grounds in Central America may serve as places where virus variants with limited circulation may be amplified upon arrival of yearly migrants. Although additional bird species need to be investigated, the high relative abundance of the bluewinged teals in comparison to other duck species in Central America, the particular behavior of this long-distance migrant (3, 34, 38), and the diversity of viruses found in these ducks makes them a candidate species for targeted IAV surveillance in the Neotropics. We recognize that surveillance in other species, characterization of viruses that circulate in resident bird populations (during and in between migrations), and incorporation of more systematic sampling methodologies, (including the use of geo-transmitters), are needed to better understand the ecology of IAV in this region; however, as we cannot longer ignore that Eurasian HPAI H5 viruses have been introduced to the western hemisphere, we think that sampling of hunted birds is a cost effective strategy that could be replicated in neighboring countries from the region that, like Guatemala, have limited resources to establish long term disease surveillance.
Acknowledgements Author Manuscript
The authors want to thank national regulatory agencies (CONAP and CECON) and licensed sport-hunters for allowing sample collection from birds in Guatemala. To Jorge Paniagua, Silvia Ramirez, Silvia Sosa, Carmen Yoc, Oscar de Leon, Adan Real, Johanna Lavigne, Diego Lopez for their assistance in sample collection and processing, data management, and administrative support. To Cheryl Nichols for English editing of this manuscript. This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) Center for Research on Influenza Pathogenesis (CRIP) contracts No. HHSN266200700010C and HHSN272201400008C.
Abbreviations AIV
Avian influenza virus
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HA (H)
hemagglutinin
NA (N)
neuraminidase
LPAIV
Low pathogenicity avian influenza virus
HPAIV
Highly pathogenic avian influenza virus
rRT-PCR
real-time reverse-transcriptase polymerase chain reaction
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Figure 1.
Temporal distribution of IAV prevalence in blue-winged teals in Guatemala, 2010-2013
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Figure 2.
Virus subtypes (HA and NA) by month obtained from wintering waterfowl in Guatemala between 2010 and 2013.
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Avian Dis. Author manuscript; available in PMC 2016 August 29. 1(50)
2
Total
102
129(23.5)
(0) 39(38.2)
2
Catoptrophorus semipalmatus (Willet)
Scolopacidae
1(50)
Fulica americana (American Coot)
Rallidae
Eudocimus albus (White Ibis)
0(0) 1(25)
Phalacrocorax brasilianus (Neotropic Cormorant)
Phalacrocoracidae
(0)
0(0)
Pelecanus erythrorhynchos (American White Pelican)
Limnodromus scolopaceus (Long-billed Dowitcher)
0(0)
Zenaida macroura (Mourning Dove)
Pelecanidae
550
1
1
4
1
1
2
148(24.7)
0(0)
0(0)
0(0)
0(0)
2(40)
8
1
1
1
2
1
1(100) 135(24.5)
0(0)
0(0)
Columba flavirostris (Red-billed Pigeon)
Columbidae
16 434
Zenaida asiatica (White-winged Dove)
0(0)
Mycteria americana (Wood Stork)
Ciconiidae
Threskiornithidae
0(0)
Positive (%)
Streptopelia decaocta (Eurasian Collared-Dove)
0(0)
(0)
2(100)
Dendrocygna bicolor (Fulvous Whistling-Duck) 2
(0)
1(50)
2
0(0)
(0)
Dendrocygna autumnalis (Black-bellied WhistlingDuck)
7
1(6.3) 99(22.8)
Cairina moschata (Muscovy Duck)
Anas sp. (Dabbling duck)
1 82
0(0)
4(57.1)
Anas discors (Blue-winged Teal)
Aythya affinis (Lesser Scaup)
0(0) 29(35.4)
Anas crecca (Green-winged Teal)
74
2
Total
8(29.6)
28(37.8)
0(0)
Positive (%)
2(28.6) 4
Total
Anas clypeata (Northern Shoveler) 1(25)
Positive (%)
598
1
5
2
1
1
550
1
27
7
3
Total
2012-2013
Anas americana (American Wigeon)
Anas acuta (Northern Pintail)
Species (Common name)
2011-2012
Oxyura jamaicensis (Ruddy Duck)
Anatidae
Family
2010-2011
316(25.3)
1(25)
0(0)
1(50)
1(25)
0(0)
0(0)
0(0)
2(15.4)
0(0)
0(0)
0(0)
0(0)
2(100)
1(50)
0(0)
0(0)
4(57.1)
263(24.7)
2(11.1)
37(35.2)
2(28.6)
0(0)
Positive (%)
Total
1250
4
1
2
4
1
1
2
13
2
1
1
2
2
2
2
2
7
1066
18
105
7
5
Total
Percentage prevalence for waterfowl species sampled during the wintering migration seasons in Guatemala, 2010-2013
Author Manuscript
Table 1 Gonzalez-Reiche et al. Page 13
Author Manuscript
Author Manuscript H5N3
Anas crecca (Green-winged teal)
Anas discors (Blue-winged teal)
1
-
Total
-
Anas clypeata (Northern shoveler)
2010
Anas americana (American wigeon)
Species
H11N3 H11N9
H4N8
21
H14N5 -
H4N6
H4N3 (2)
21
H4N2 (4)
H4N2 (2)
H14N3 (7)
H12N5 H14N3 (2)
H3N8
12
H11N3
H5N3
H3N3 (2)
H1N3 (3)
H3N3
H3N8 (2)
H14N6
-
H11N3
-
2011
H3N2 (10)
-
-
-
-
2010
H3N2 (4)
-
-
-
2012
December
H1N3 (3)
-
H1N3
-
2011
November
5
H4N2
H3N2
H2N3
-
H4N2
H4N2
2012
1
-
-
H7N3
-
2012
7
H5N3
H14N4 (2)
H14N3 (2)
H7N9
H3N8
-
2013
January
Author Manuscript
Subtypes of IAV isolated during the wintering season in Guatemala, 2010-2013
68
61
1
5
1
Total
Author Manuscript
Table 2 Gonzalez-Reiche et al. Page 14
Avian Dis. Author manuscript; available in PMC 2016 August 29.
